Chediak-Higashi (CHS) is a rare childhood autosomal recessive disorder for which the primary defect is unknown. The aim of the present study is to contribute information to the delineation of the molecular defect at the protein level while also using the disease as a model for the examination of lysosomal function which seems to be impaired in CHS. Our experimental approach will involve a detailed biochemical characterization of lysosomal enzymes and lysosomal membranes from cultured CHS human fibroblasts and where necessary cultured fibroblasts and macrophages from the beige mouse model for the disease. Among the specific experimental paradigms that will be used is a free-flow electrophoresis procedure to isolate lysosomal organelles and their enzyme contents from lysed normal and CHS cells. Selected biochemical properties of each sample will be subsequently examined to ascertain the reason(s) for the reduced lysosomal enzyme activities and impaired lysosomal function. These studies will focus on the binding and elution of lysosomal enzymes from specific lectins including concanavalin A before and after endo H treatment, Ricinus communis, and wheat germ agglutinin; isoelectric focusing profiles of lysosomal enzymes before and after neuraminidase treatment; and receptor-mediated pinocytosis of the partially purified N-acety-Beta-D-hexosaminidase before and after separate incubations with endo H and alkaline phosphatase. In addition, the CHS and normal lysosomal membranes will be characterized with respect to the properties of the phosphomannosyl receptor and their protein and glycoprotein compositions. Furthermore, the use of radiolabeled sucrose-LDL conjugated may allow us to assess whether the CHS mutation affects the molecular mechanism of receptor-mediated endocytosis, endosome-lysosome fusion, degradation and permeability of the resulting products to lysosomal and cellular membranes.